RESUMEN
Blood-vessel formation generates unique vascular patterns in each individual. The principles governing the apparent stochasticity of this process remain to be elucidated. Using mathematical methods, we find that the transition between two fundamental vascular morphogenetic programs-sprouting angiogenesis and vascular remodeling-is established by a shift of collective front-to-rear polarity of endothelial cells in the mouse retina. We demonstrate that the competition between biochemical (VEGFA) and mechanical (blood-flow-induced shear stress) cues controls this collective polarity shift. Shear stress increases tension at focal adhesions overriding VEGFA-driven collective polarization, which relies on tension at adherens junctions. We propose that vascular morphogenetic cues compete to regulate individual cell polarity and migration through tension shifts that translates into tissue-level emergent behaviors, ultimately leading to uniquely organized vascular patterns.
Asunto(s)
Polaridad Celular , Células Endoteliales , Uniones Adherentes/metabolismo , Animales , Movimiento Celular/fisiología , Polaridad Celular/fisiología , Células Endoteliales/metabolismo , Ratones , Morfogénesis , Retina/metabolismoRESUMEN
Nuclear position is central to cell polarization, and its disruption is associated with various pathologies. The nucleus is moved away from the leading edge of migrating cells through its connection to moving dorsal actin cables, and the absence of connections to immobile ventral stress fibers. It is unclear how these asymmetric nucleo-cytoskeleton connections are established. Here, using an in vitro wound assay, we find that remodeling of endoplasmic reticulum (ER) impacts nuclear positioning through the formation of a barrier that shields immobile ventral stress fibers. The remodeling of ER and perinuclear ER accumulation is mediated by the ER shaping protein Climp-63. Furthermore, ectopic recruitment of the ER to stress fibers restores nuclear positioning in the absence of Climp-63. Our findings suggest that the ER mediates asymmetric nucleo-cytoskeleton connections to position the nucleus.
Asunto(s)
Actinas , Retículo Endoplásmico , Actinas/metabolismo , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Retículo Endoplásmico/metabolismo , Fibras de Estrés/metabolismoRESUMEN
Skeletal muscle myofibers are large and elongated cells with multiple and evenly distributed nuclei. Nuclear distribution suggests that each nucleus influences a specific compartment within the myofiber and implies a functional role for nuclear positioning. Compartmentalization of specific mRNAs and proteins has been reported at the neuromuscular and myotendinous junctions, but mRNA distribution in non-specialized regions of the myofibers remains largely unexplored. We report that the bulk of mRNAs are enriched around the nucleus of origin and that this perinuclear accumulation depends on recently transcribed mRNAs. Surprisingly, mRNAs encoding large proteins - giant mRNAs - are spread throughout the cell and do not exhibit perinuclear accumulation. Furthermore, by expressing exogenous transcripts with different sizes we found that size contributes to mRNA spreading independently of mRNA sequence. Both these mRNA distribution patterns depend on microtubules and are independent of nuclear dispersion, mRNA expression level and stability, and the characteristics of the encoded protein. Thus, we propose that mRNA distribution in non-specialized regions of skeletal muscle is size selective to ensure cellular compartmentalization and simultaneous long-range distribution of giant mRNAs.
Asunto(s)
Fibras Musculares Esqueléticas , Músculo Esquelético , Núcleo Celular/genética , ARN Mensajero/genética , TendonesRESUMEN
Despite promising preclinical results, average response rates to anti-VEGF therapies, such as bevacizumab, are reduced for most cancers, while incurring in remarkable costs and side effects. Currently, there are no biomarkers available to select patients that can benefit from this therapy. Depending on the individual tumor, anti-VEGF therapies can either block or promote metastasis. In this context, an assay able to predict individual responses prior to treatment, including the impact on metastasis would prove of great value to guide treatment options. Here we show that zebrafish xenografts are able to reveal different responses to bevacizumab in just 4 days, evaluating not only individual tumor responses but also the impact on angiogenesis and micrometastasis. Importantly, we perform proof-of-concept experiments where clinical responses in patients were compared with their matching zebrafish Patient-Derived Xenografts - zAvatars, opening the possibility of using the zebrafish model to screen bevacizumab therapy in a personalized manner.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Bevacizumab/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Neovascularización Patológica/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Apoptosis , Proliferación Celular , Femenino , Humanos , Metástasis de la Neoplasia , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Pez CebraRESUMEN
Morphogenesis of hierarchical vascular networks depends on the integration of multiple biomechanical signals by endothelial cells, the cells lining the interior of blood vessels. Expansion of vascular networks arises through sprouting angiogenesis, a process involving extensive cell rearrangements and collective cell migration. Yet, the mechanisms controlling angiogenic collective behavior remain poorly understood. Here, we show this collective cell behavior is regulated by non-canonical Wnt signaling. We identify that Wnt5a specifically activates Cdc42 at cell junctions downstream of ROR2 to reinforce coupling between adherens junctions and the actin cytoskeleton. We show that Wnt5a signaling stabilizes vinculin binding to alpha-catenin, and abrogation of vinculin in vivo and in vitro leads to uncoordinated polarity and deficient sprouting angiogenesis in Mus musculus. Our findings highlight how non-canonical Wnt signaling coordinates collective cell behavior during vascular morphogenesis by fine-tuning junctional mechanocoupling between endothelial cells.
Asunto(s)
Movimiento Celular , Células Endoteliales/fisiología , Neovascularización Fisiológica , Vía de Señalización Wnt , Proteína Wnt-5a/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Animales , Ratones , Unión Proteica , Vinculina/metabolismo , alfa Catenina/metabolismoRESUMEN
Cell migration is essential during development, regeneration, homeostasis, and disease. Depending on the microenvironment, cells use different mechanisms to migrate. Yet, all modes of migration require the establishment of an intracellular front-rear polarity axis for directional movement. Although front-rear polarity can be easily identified in in vitro conditions, its assessment in vivo by live-imaging is challenging due to tissue complexity and lack of reliable markers. Here, we describe a novel and unique double fluorescent reporter mouse line to study front-rear cell polarity in living tissues, called GNrep. This mouse line simultaneously labels Golgi complexes and nuclei allowing the assignment of a nucleus-to-Golgi axis to each cell, which functions as a readout for cell front-rear polarity. As a proof-of-principle, we validated the efficiency of the GNrep line using an endothelial-specific Cre mouse line. We show that the GNrep labels the nucleus and the Golgi apparatus of endothelial cells with very high efficiency and high specificity. Importantly, the features of fluorescent intensity and localization for both mCherry and eGFP fluorescent intensity and localization allow automated segmentation and assignment of polarity vectors in complex tissues, making GNrep a great tool to study cell behavior in large-scale automated analyses. Altogether, the GNrep mouse line, in combination with different Cre recombinase lines, is a novel and unique tool to study of front-rear polarity in mice, both in fixed tissues or in intravital live imaging. This new line will be instrumental to understand cell migration and polarity in development, homeostasis, and disease.
Asunto(s)
Polaridad Celular/fisiología , Ingeniería de Proteínas/métodos , Animales , Movimiento Celular/fisiología , Núcleo Celular/metabolismo , Polaridad Celular/genética , Células Endoteliales , Colorantes Fluorescentes , Genes Reporteros , Aparato de Golgi/metabolismo , RatonesRESUMEN
In this article, we present PolNet, an open-source software tool for the study of blood flow and cell-level biological activity during vessel morphogenesis. We provide an image acquisition, segmentation, and analysis protocol to quantify endothelial cell polarity in entire in vivo vascular networks. In combination, we use computational fluid dynamics to characterize the hemodynamics of the vascular networks under study. The tool enables, to our knowledge for the first time, a network-level analysis of polarity and flow for individual endothelial cells. To date, PolNet has proven invaluable for the study of endothelial cell polarization and migration during vascular patterning, as demonstrated by two recent publications. Additionally, the tool can be easily extended to correlate blood flow with other experimental observations at the cellular/molecular level. We release the source code of our tool under the Lesser General Public License.
Asunto(s)
Polaridad Celular , Hemodinámica , Modelos Biológicos , Programas Informáticos , Remodelación VascularRESUMEN
Research on trypanosomes as a model organism has provided a substantial contribution to a detailed understanding of basic cellular processes within the last few years. At the same time, major advances in super-resolution microscopy have been achieved, facilitating the resolution of biological structures in living cells at a scale of a few nm. However, the motility of trypanosomes has prevented access to high resolution microscopy of live cells. Here, we present a hydrogel based on poly(ethylene glycol) functionalized with either norbornene or thiol moieties for UV induced thiol-ene crosslinking for the embedding and imaging of live trypanosomes. The resulting gel exhibits low autofluorescence properties, immobilizes the cells efficiently on the nanometer scale and is compatible with cell viability for up to one hour at 24 °C. We applied super-resolution imaging to the inner plasma membrane leaflet using lipid-anchored eYFP as a probe. We find specific domains within the membrane where the fluorescence either accumulates or appears diluted rather than being homogenously distributed. Based on a Ripley's analysis, the size of the domains was determined to be raccumulated=170±5 nm and rdilute>115±15 nm. We hypothesize that this structuring of the membrane is associated with the underlying cytoskeleton.
Asunto(s)
Trypanosoma brucei brucei/ultraestructura , Antígenos de Protozoos/química , Antígenos de Protozoos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biomarcadores/química , Biomarcadores/metabolismo , Membrana Celular/ultraestructura , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Hidrogeles , Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/genéticaRESUMEN
The current view of the pluripotent state is that of a transient, dynamic state, maintained by the balance between opposing cues. Understanding how this dynamic state is established in pluripotent cells and how it relates to gene expression is essential to obtain a more detailed description of the pluripotent state.In this chapter, we describe how to study the dynamic expression of a core pluripotency gene regulator-Nanog-by exploiting single-cell time-lapse imaging of a reporter mESC line grown in different cell culture media. We further describe an automated image analysis method and discuss how to extract information from the generated quantitative time-course data.
Asunto(s)
Citometría de Flujo/métodos , Proteínas de Homeodominio/análisis , Microscopía Confocal/métodos , Células Madre Embrionarias de Ratones/citología , Análisis de la Célula Individual/métodos , Animales , Técnicas de Cultivo de Célula/métodos , Ciclo Celular , Línea Celular , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Proteína Homeótica Nanog , Imagen Óptica/métodos , Factores de TiempoRESUMEN
The plasma membrane is a highly complex, organized structure where the lateral organization of signaling proteins is tightly regulated. In the case of Ras proteins, it has been suggested that the differential activity of the various isoforms is due to protein localization in separate membrane compartments. To date, direct visualization of such compartmentalization has been achieved only by electron microscopy on membrane sheets. Here, we combine photoactivated light microscopy with quantitative statistical analysis to visualize protein distribution in intact cells. In particular, we focus on the localization of HRas and its minimal anchoring domain, CAAX. We demonstrate the existence of a complex partitioning behavior, where small domains coexist with larger ones. The protein content in these domains varied from two molecules to tens of molecules. We found that 40% of CAAX and 60% of HRas were localized in domains. Subsequently, we were able to manipulate protein distributions by inducing coalescence of supposedly cholesterol-enriched domains. Clustering resulted in an increase of the localized fraction by 15%.
Asunto(s)
Membrana Celular/ultraestructura , Fibroblastos/ultraestructura , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Células 3T3 , Animales , Membrana Celular/metabolismo , Proteínas de la Membrana/química , Ratones , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas p21(ras)/químicaRESUMEN
Here we report on a method to track individual molecules on nanometer length and microsecond timescales using an optical microscope. Our method is based on double-labeling of a molecule with two spectrally distinct fluorophores and illuminating it with laser pulses of different wavelengths that partially overlap temporally. We demonstrate our method by using it to resolve the motion of short DNA oligomers in solution down to a timescale of 100 µs.